Analysis of correspondence between Q-banding pattern of the chicken mitotic chromosomes and the chromomere pattern of lampbrush chromosomes by FISH and fluorochrome stainingS. Galkina, N. Lukina, E. Vasilieva, L.Andreozzi, C. Federico, S. Saccone, G. Bernardi, A. Rodionov
Biological Institute of St.Petersburg University, Russia; University of Catania, Italy; University of Bologna, Italy; Stazione Zoologica, Napoli, Italy
Fig. 1.Enlarge in new window (128K)
The characteristic feature of the eucaryotic mitotic chromosome is their distinct longitudinal differentiation exhibiting by chromosome banding pattern. On the other hand, in meiotic prophase the chromosome becomes visibly folded in chromomeres and loops. Most studies on correspondence analysis between banding pattern of metaphase chromosome and chromomere pattern of meiotic prophase chromosome employed pachytene and diakinetic chromosomes from mammalian spermatocytes (e.g. Bordjadze, Prokofieva-Belgovskaia, 1971; Pathak et al., 1976; Jagiello, Fang, 1982; Fang, Jagiello, 1981, 1988; Luciani et al., 1975, 1988). We attempted to determine the correspondence between G(Q) banding pattern of the chicken mitotic chromosomes and the loop/chromomere pattern of the lampbrush chromosomes from growing oocytes using the lampbrush chromosome from bird (chicken) oocytes.
Chicken lampbrush chromosomes (LBCs) are giant diplotene bivalents which are about 33 times more long than mitotic chromosomes (Fig. 1). The axis of each homologue consists of a lot of chromomeres carrying lateral loops. There are two main types of the lampbrush chromosome Markers: lateral loops with distinctive appearances (e.g. TBL11, PBL11) and marker chromomeres (e.g. C1I21, C1C51).
LBCs were stained with the fluorochromes, both AT-specific DAPI and GC-specific chromomycin A3 (CMA). After DAPI/CMA-staining (Ambros, Sumner, 1987) we observed bright CMA-positive yellow-green fluorescence of the lateral loops and DAPI-specific blue fluorescence of the chromomeres (Fig. 2). Each chicken macrobivalent exhibits a characteristic DAPI-positive chromomere pattern. On the contrary to amphibian LBCs, the chromomeres were irregularly distributed along the chicken LBC axes.
As a rule, there are few groups of bright chromomeres, some separate brilliant chromomeres, and chromomere regions that are comparatively dim. In particular, bright chromomere clusters present in the regions J1, 1J2, 1L1 of the 1q-arm (Fig. 2). There are also few brilliant chromomeres on the 1q (C1I21, C1I11, C1I12, C1G1/LPBL11), few bright regions (1C1, 1B) and a single brilliant chromomere C1C51 in the 1p-arm. Subtelomeric regions of the chicken macrobivalent fluoresce as green-yellow (CMA-positive).
Fig. 2.Enlarge in new window (58K)
A ideogram of chicken mitotic chromosome 1
B mitotic chromosome 1 stained by DAPI/CMA
C lampbrush chromosome 1 stained by DAPI/CMA
The DAPI/CMA staining pattern of the chicken lampbrush microchromosomes (microLBCs) is very specific (Fig. 3). All of them carry a bright chromomere near one of the telomeres, all other chromomeres fluoresce dully after DAPI but comparatively bright after CMA-staining. It seems that the single DAPI-bright chromomere of the microchomosomes is the centromeric heterochromatic band.
It appeared reasonable to assume that the regions of the DAPI-positive chromomeres of the chicken macroLBCs correspond to AT-enriched G(Q-positive)-bands of the chicken mitotic chromosomes. On the other hand, the CMA-positive regions correspond to the GC-enriched R(Q-negative)-bands.
Fig. 3.Enlarge in new window (50K)
Lampbrush microchromosomes stained by DAPI, CMA and after FISH with GC-enriched isochore fraction.
To check this assumption we have compared the chicken mitotic and lampbrush chromosomes by FISH with GC-enriched chicken isochore families. On the chicken mitotic chromosomes the DOP-PCR generated probes (Telenius et al., 1992) of the GC-richest isochores were strongly hybridized with many microchromosomes. Some internal R(Q-negative)-bands and almost all telomeric segments of macrochromosomes were also labelled (Fig. 4).
The hybridization signals observed with the GC-rich isochore fraction on the meiotic lampbrush macrochromosome 1 distributed in telomeric and internal regions. The strongest signal was on the telomeric bow-like loops (TBL11). The labelling of the internal regions was non-uniform. There were strong signals on few loops (e.g. PBL11) and weakly labelled regions along the arms that appeared to be corresponding to some R-bands of the chicken mitotic chromosomes. Fig. 4 shows that there are few strong labeled regions of the 1q-arm in both mitotic and lampbrush chromosomes.
At least 15 microchromosomes showed strong signals on their lateral loops (Fig. 3). There were few types of microbivalents: 1) microLBCs with pericentromeric labelled loops that are situated near DAPI-positive chromomere; 2) microLBCs with telomeric labelled loops; 3) microLBCs that carry GC-enriched isochore positive loops along all their length. There were some microbivalents without hybridization signals also.
Fig. 4.Enlarge in new window (57K)
Mitotic and lampbrush chromosome 1 after FISH with GC-enriched isochore fraction. Green signals on the PI-stained mitotic chromosome is due to FITC; red signals on the CMA-stained LBC is due to Cy3.
Figure 5 shows the correspondence between the Q-banding pattern of chicken mitotic chromosome 1 and the loop-chromomere pattern of the meiotic diplotene lampbrush chromosome. One can see that there is good correlation between metaphase chromo- some Q-positive bands and clusters of lampbrush bright chromomeres. Q-negative bands is equal to the regions of chicken lampbrushes, consisting Mainly of the ďAT/GC-neutral" chromomeres. Red regions on both LBC and mitotic chromosome exhibit heavy isochore-positive sites.
Fig. 5.Enlarge in new window (24K)
Acknowledgements. This work was supported by the INTAS (grant 97-1610), the Russian Basic Research Foundation (grant #00-04-49327 and #98-04-49829). Authors thank Prof. E.R.Gaginskaya of the St.Petersburg University, Microscopic Research Center "Chromas" (RBRF #00-04-55013) and the Ministry of Science and Technology Research Program "Comus-1" (#06-03) for technical supporting.